Treatment of streptococcal infections

ABSTRACT

Immunogenic compositions and methods of treating a  Streptococcus pneumoniae  infection are described herein.

The present application relates to the field of vaccines and immunogeniccompositions that protect against Streptococcal disease and particularlyto methods of treating Streptococcus pneumoniae disease using vaccinescontaining proteins from the polyhistidine triad family of proteins, andin particular, to methods of treatment using these vaccines andimmunogenic compositions

BACKGROUND

Streptococcus pneumoniae is one of the leading causes of infectiousmorbidity and mortality in the world, responsible for a large spectrumof infections such as otitis media, pneumonia, bacteremia and meningitis{Hausdorff 2005, McCullers 2001}. The emergence of antibiotic-resistantstrains of this micro-organism has further underlined the need forproviding effective prophylactic vaccination {Lynch, III 2005,Bridy-Pappas 2005}.

Current vaccines are composed of epidemiologically dominantserotype-based selections of pneumococcal capsular polysaccharides,conjugated or not to a carrier protein {Dagan 2004, Fedson 2004, Mbelle1999, Smart 1987}. However, the vaccine formulations do not cover allserotypes of this micro-organism, which might particularly be ofrelevance in certain regions of the globe with different dominantserotypes {Dagan 1992}. In addition, one may expect that the use ofserotype-specific vaccines could allow at term the positive selection ofnon-vaccine serotypes {Nunes 2008, Singleton 2007}.

An alternative approach involves the development of vaccines that targetcommon pneumococcal antigens. Among multiple candidates, the Pht proteinfamily, restricted to the genus Streptococcus, comprises promising ones,being well-conserved across the pneumococcal species {Hamel 2004, Zhang2001}, and being antibody targets in infected individuals and protectiveupon challenge in immunized mice {Beghetto 2006}. Originally, thisprotein family was independently reported by three groups, and threeseparate denominations were used: Pht (for pneumococcal histidine triad){Adamou 2001}, Php (for pneumococcal histidine protein) {Zhang 2001},and BVH {Hamel 2004}. Those proteins are characterized by a histidinetriad motif, HxxHxH, repeated five to six times in their amino-acidsequences. Four members of this family have been described: PhtA(BVH-11-3), PhtB (PhpA/BVH-11) and PhtD (BVH-11-2) that share up to 81%sequence identity, and PhtE (BVH-3) that diverges from the three otherproteins, showing only up to 35% identity with them. It is a longerprotein, the only one with six repeats of the histidine triad motif. Inmouse immunization studies, all members of the Pht family have beenshown to afford a high level of protection to subsequent pneumococcalinfection with a number of different strains/serotypes {Adamou 2001,Hamel 2004, Ogunniyi 2007, Wizemann 2001, Zhang 2001}.

Despite their potential importance in vaccination against S. pneumoniae,the biological function of these proteins has yet to be determined.Results from antibody-labeling and flow cytometry experimentsdemonstrated that the Pht proteins are exposed on the surface of theencapsulated bacterium {Hamel 2004}, which is in agreement with theirrelevance as vaccine target. By signature-tagged mutagenesis, it hasbeen suggested that PhtA, PhtB, and PhtD are involved in lung-specificvirulence {Hava 2002}, without further indication about their biologicalfunction. Among their putative roles, neutralization of the complementfactor C3b has been suggested {Hostetter 1999, Ogunniyi 2009}, whichimplies that they would interfere with phagocytosis. Besides that, arole in adherence is also suspected. Indeed, a genetic link between phtDand lmb, the latter encoding a putative laminin adhesion protein{Spellerberg 1999}, has been reported {Panina 2003}. At last, due to thehigh number of histidine residues in the histidine triads, it has beensuggested that the Pht proteins may be involved in DNA and/or metalbinding {Adamou 2001}. More specifically, some studies highlighted alink between the Pht family and zinc. Indeed, AdcR-binding sites havebeen found in the upstream regions of the pht genes, AdcR beingdescribed as a transcription factor that regulates zinc uptake {Panina2003}. Furthermore, the crystal structure of a portion of PhtA revealedthe presence of zinc ions bound to a histidine triad domain{Riboldi-Tunnicliffe 2005}. It is not clear, however, whether zincscavenging or transport is the function of those proteins, or whetherzinc rather plays a conformational or functional role.

Important aspects that need to be addressed for vaccine candidates aretheir level of expression and associated regulation, their occurrence aswell as their sequence variability. Therefore, we have addressed thesedifferent aspects with regard to the Pht proteins.

Streptococcus pneumoniae elicits different disease states exhibitingdifferent pathologies depending on the site at which the pneumococcalpopulation expands. Septicaemia occurs where the S. pneumoniae enters toblood steam, whereas pneumonia occurs where S. pneumoniae multiplies inthe lung. S. pneumoniae is also an important pathogen in otitis mediainfections. S. pneumoniae can also enter the cerebrospinal fluid tocause meningitis.

There is a need to develop better pneumococcal vaccines which are ableto target specific pneumococcal diseases and provide optimal protectionagainst a particular form of pneumococcal disease.

Accordingly there is provided a method of treating or preventingStreptococcus pneumoniae infection wherein the Streptococcus pneumoniaeinfection occurs in an environment where the concentration of Zn²⁺and/or Mn²⁺ is sufficiently low to upregulate the expression of at leastone PhtX protein in the Streptococcus pneumoniae; comprising the step ofadministering a pharmaceutically effective amount of the PhtX protein toa human patient.

In a second aspect of the invention there is provided an immunogeniccomposition comprising a pharmaceutically effective amount of anisolated PhtX protein for use in the treatment or prevention of aStreptococcus pneumoniae infection wherein the Streptococcus pneumoniaeinfection occurs in a human patient in an environment where theconcentration of Zn²⁺ and/or Mn²⁺ is sufficiently low to upregulate theexpression of at least one PhtX protein in the Streptococcus pneumoniae.

In a third aspect of the invention there is provided a use of apharmaceutically effective amount of an isolated PhtX protein in themanufacture of a medicament for the treatment or prevention of aStreptococcus pneumoniae infection wherein the Streptococcus pneumoniaeinfection occurs in a human patient in an environment where theconcentration of Zn²⁺ and/or Mn²⁺ is sufficiently low to upregulate theexpression of at least one PhtX protein in the Streptococcus pneumoniae.

FIGURE LEGENDS

FIG. 1. Organization of the pht genes in Streptococcus pneumoniaeserotype 4 strain TIGR4.

FIG. 2. Promoter-containing upstream regions of the pht genes. (a) phtEgene, (b) phtA gene, (c) phtB gene, (d) lmb gene, (e) yfnA gene. The −35and −10 regions are double-underlined, transcription start sites areindicated by a boldface letter and the symbol (+1), putative ribosomebinding sites (rbs) are underlined, and open reading frames arerepresented by arrows indicating the direction of transcription over aseries of boldface letters. The numbers on the left correspond tosequence positions in GenBank accession numbers AY569979 (a, d and e)and AY569980 (b and c).

FIG. 3. Rho-independent transcription terminator sequences of the phtand ptsl genes. (a) phtE, (b) phtB, (c) phtD, (d) phtA and (e) pstlgenes. Stop codons are underlined in boldface, terminator regions areunderlined, and sequences underlined with a discontinuous line indicatethe hairpin region of the terminators. Open reading frames arerepresented by arrows indicating the direction of transcription over aseries of boldface letters. In (a), the region in italics (phtF gene;putative start codon doubly underlined) presents 78% identity with thefirst 481 bp of the phtE gene. However, underlined stop codons preventsignificant gene translation. The numbers correspond to sequencepositions in GenBank accession numbers AY569979 (a and c) and AY569980(b, d and e).

FIG. 4. RT-PCR analyses of the pht transcripts. (a) 1% agarose gelshowing the RT-PCR products with template RNA from cells grown tomid-log growth phase. Lanes 1 to 8 correspond to regions 1 to 8 in theschematic representation in (b). The RT-PCR products shown in lanes 1 to8 were generated using primer pairs that flanked the correspondingregions depicted in the scheme. The length of each predicted RT-PCRproduct is indicated in parentheses.

FIG. 5. SDS-PAGE immunoblotting of bacterial extracts. Anti-PhtDantibody was used to probe extracts from the PhtABDE⁻ quadruple mutant(A), the PhtE⁻ mutant (B), PhtD⁻ mutant (C), PhtB⁻ mutant (D), PhtA⁻mutant (E), and the wild-type (F) strains. The position of the differentPht bands is indicated on the right, and a molecular mass mark is on theleft side of the picture.

FIG. 6. Growth curves of 4/CDC wild-type strain and Pht-deficientmutants in MS medium (a). The growth curves of the wild-type,PhtD-deficient and Pht quadruple mutant were also determined in MS withor without Zn²⁺ 200 μM (b), Mn²⁺ 200 μM (c), or Fe²⁺ 200 μM (d). Eachfigure depicts the results of one experiment representative of three.

FIG. 7. WU2 bacterial cells were cultured with or without TPEN 30 μM, azinc chelator. Next, cells were probed with anti-PhtB/D (a), anti-PhtE(b), anti-PhtD/E (c), or anti-type 3 polysaccharide (d) antibodiesfollowed by AlexaFluor-conjugated goat anti-mouse secondary antibodybefore they were analyzed by flow cytometry. As controls, cells wereincubated with the secondary conjugate antibody. Representative FACSplots of the different conditions are shown.

FIG. 8. Western blot analysis. Whole-cell extracts were submitted toSDS-PAGE followed by immunoblotting. Nine different strains were probedwith a polyclonal anti-PhtD (a), and 8 with a polyclonal anti-PhtE (b).Molecular mass marker is shown.

FIG. 9. Signal sequences comparison of PhtX family members. The shadedareas identify amino acids which are conserved in at least ⅔ PhtX familymembers.

FIG. 10. Mice survival upon lethal S. pneumoniae intranasal challenge.Mice (n=20/group) were immunized with AS02-adjuvanted PhtD, PhtA, PhtB,PhtE or AS02 alone (control) before they were challenged with the type3/43 pneumococcal strain. Statistical analyses were carried out with thelogrank test, compared with control: PhtD, p=0.0126; PhtA, p=0.0103;PhtB, p=0.0038; PhtE, p=0.0033.

FIG. 11. Antibody levels after immunization. A.) Mice were immunizedsystemically with AS02-adjuvanted CbpA, PspA, or PhtD. B) Mice wereimmunized intra-nasally with LT-adjuvanted CbpA, PspA or PhtD. In bothcases, blood was taken on day 42, and the levels of specific antibodieswere measured by ELISA.

FIG. 12. Mice survival upon lethal S. pneumoniae intranasal challenge.Mice were immunized with AS02-adjuvanted CbpA, PspA, PhtD or AS02 alone(control) before they were challenged with the type 2/D39 (A), type 3/43(B), or type 4/CDC (C) pneumococcal strains. Statistical analyses werecarried out with the logrank test, compared with control: (A) CbpA,p=0.0002; PspA, p=0.0001; PhtD, p=0.0009. (B) CbpA, p=0.885; PspA,p=0.184; PhtD, p=0.027. (C) CbpA, p=0.825; PspA, p=0.538; PhtD,p<0.0001.

FIG. 13. Vaccine efficacy in a S. pneumoniae naso-pharyngealcolonization model. Balb/c mice were immunized with PhtD, PhtA, PhtB,PhtE, or LT alone (Ctrl), before they were intranasally challenged withthe 2/D39 pneumococcal strain. Bacterial colonies were counted in nasalwashings at day 2 and at day 6 post-challenge, and expressed as log 10mean cfu. Each dot represents a mouse. Black horizontal bars aregeometric means. Dashed line indicates limit of detection (at 0.84).Statistical analyses were carried out per day with ANOVA. Allsignificant differences, compared with control, are shown. * p<0.05; ns:not significant.

FIG. 14. Vaccine efficacy in a S. pneumoniae naso-pharyngealcolonization model. Balb/c mice were immunized with either CbpA, PspA,PhtD, PsaA, or LT alone (Control), before they were intranasallychallenged with either the 2/D39 (A), the 4/CDC (B), or the 6B/CDC (C)pneumococcal strain. Bacterial colonies were counted in nasal washingsat day 2 and at day 6 post-challenge, and expressed as log 10 mean cfu.Each dot represents a mouse. Dashed lines indicate limit of detection(at 0.84). Black horizontal bars are geometric means. Statisticalanalyses were carried out per day with ANOVA. All significantdifferences, compared with control, are shown. * p<0.05; ** p<0.01;***p<0.001, ns: not significant.

FIG. 15. Vaccine efficacy in a S. pneumoniae lung colonization model.CBA/J mice were immunized with AS02-adjuvanted PhtD or with AS02 only(Ctrl), before they were challenged with the moderately virulent19F/2737 pneumococcal strain. Lungs were taken at day 3, 4 or 5post-challenge, and bacterial load was evaluated by colony counting(cfu). Each dot represents a mouse. Dashed line indicates limit ofdetection (at 2). Black horizontal bars are geometric means. The groupswere compared with ANOVA2 over the three days, followed by Tuckey-HSD:p<0.0001.

DETAILED DESCRIPTION

The invention provides a method of treating or preventing Streptococcuspneumoniae infection wherein the Streptococcus pneumoniae infectionoccurs in an environment where the concentration of Zn²⁺ and/or Mn²⁺ issufficiently low to upregulate the expression of at least one PhtXprotein in the Streptococcus pneumoniae; comprising the step ofadministering a pharmaceutically effective amount of the PhtX protein toa human patient.

Zn2+ and Mn2+ are present in a human body in both free and bound forms.Bound Zn2+ or Mn2+ is bound to proteins such as albumin and makes up themajority of these ions. On the other hand, a small amount of free Zn2+or Mn2+ is present in body fluids such as blood, lymph, interstitialfluid or cerebrospinal fluid. The term “bound” relates to ions which aretightly associated with proteins such as albumin. The term “free”relates to ions which are not tightly associated with proteins such asalbumin. Such free ions are more available for uptake by S. pneumoniae.In an embodiment, the method of the invention provides a method oftreating or preventing Streptococcus pneumoniae infection wherein theStreptococcus pneumoniae infection occurs in an environment where thefree concentration of Zn²⁺ and/or Mn²⁺ is sufficiently low to upregulatethe expression of at least one PhtX protein in the Streptococcuspneumoniae. In an embodiment, the method of the invention provides amethod of treating or preventing Streptococcus pneumoniae infectionwherein the Streptococcus pneumoniae infection occurs in an environmentwhere the bound and/or free concentration of Zn²⁺ and/or Mn²⁺ issufficiently low to upregulate the expression of at least one PhtXprotein in the Streptococcus pneumoniae.

Bt the term “sufficiently low to upregulate the expression of at leastone PhtX protein” for the purposes of the invention, it is meant thatthe level of Zn2+ and/or Mn2+ (bound and/or free) is:

-   -   a) lower than that usually found in the equivalent position of a        human body, such that the level of expression of at least one        PhtX protein in S. pneumoniae present in that body, is higher        than the level of expression of PhtX in S. pneumoniae found in        the equivalent compartment of the body under normal conditions        (i.e. in an individual with average Zn2+ or Mn2+ levels); or    -   b) lower than that found in regions of high Zn2+ availability of        the body such that the level of expression of at least one PhtX        protein in S. pneumoniae present in that location, is higher        than the level of expression of PhtX in S. pneumoniae found in        the region of high Zn2+ availability of the body in the same        individual.

In an embodiment, the level of bound Zn2+ is reduced. In an embodiment,the level of free Zn2+ is reduced.

Situation a) may be achieved through a decrease in the overall levels ofZn2+ and/or Mn2+ whereas situation b) may be achieved by the S.pneumoniae infection occurring at a site which has comparatively lowlevels of Zn2+ and/or Mn2+.

A PhtX protein is a member of the histidine triad family of proteins.The PhtX protein is optionally the full length protein but may be afragment of the protein or a fragment or fusion protein comprising atleast one fragment or full length PhtX protein. The PhtX proteinexpressed in S. pneumoniae will be a full length protein, however thePhtX protein administered to a human patient is optionally a full lengthPhtX protein, a fragment of a PhtX protein or a fusion proteincomprising at least one PhtX protein or fragment thereof.

In an embodiment, the PhtX protein is selected from the group consistingof PhtA, PhtB, PhtD and PhtE. In an embodiment, the PhtX protein isPhtD.

The present invention relates to members of the polyhistidine triadfamily (Pht) proteins, fragments or fusion proteins thereof. The PhtA,PhtB, PhtD or PhtE proteins may have an amino acid sequence sharing 80%,85%, 90%, 95%, 98%, 99% or 100% identity with a sequence disclosed in WO00/37105 or WO 00/39299 (e.g. with amino acid sequence 1-838 or 21-838of SEQ ID NO: 4 of WO 00/37105 for PhtD).

The Pht (Poly Histidine Triad) family comprises proteins PhtA, PhtB,PhtD, and PhtE. The family is characterized by a lipidation sequence,two domains separated by a proline-rich region and several histidinetriads, possibly involved in metal or nucleoside binding or enzymaticactivity, (3-5) coiled-coil regions, a conserved N-terminus and aheterogeneous C terminus. It is present in all strains of pneumococcitested. Homologous proteins have also been found in other Streptococciand Neisseria. It is understood, however, that the terms Pht A, B, D,and E refer to proteins having sequences disclosed in the citationsabove or below as well as naturally-occurring (and man-made) variantsthereof that have a sequence homology that is at least 90% identical tothe referenced proteins. Optionally it is at least 95% identical or atleast 97% identical.

With regards to the PhtX proteins, PhtA is disclosed in WO 98/18930, andis also referred to Sp36. As noted above, it is a protein from thepolyhistidine triad family and has the type II signal motif of LXXC.PhtD is disclosed in WO 00/37105, and is also referred to Sp036D. Asnoted above, it also is a protein from the polyhistidine triad familyand has the type II LXXC signal motif. PhtB is disclosed in WO 00/37105,and is also referred to Sp036B. Another member of the PhtB family is theC3-Degrading Polypeptide, as disclosed in WO 00/17370. This protein alsois from the polyhistidine triad family and has the type II LXXC signalmotif. For example, an immunologically functional equivalent is theprotein Sp42 disclosed in WO 98/18930. A PhtB truncate (approximately 79kD) is disclosed in WO99/15675 which is also considered a member of thePhtX family. PhtE is disclosed in WO00/30299 and is referred to asBVH-3. Where any Pht protein is referred to herein, it is meant thatimmunogenic fragments or fusions thereof of the Pht protein can be used.For example, a reference to PhtX includes immunogenic fragments orfusions thereof from any Pht protein. A reference to PhtD or PhtB isalso a reference to PhtDE or PhtBE fusions as found, for example, inWO0198334.

The method of treatment or use of the invention may involve theadministration of the full length PhtX protein, a fragment of the PhtXprotein or a fusion protein containing at least 1 or 2 fragment(s) ofPhtX proteins. Where fragments of Pht proteins are used (separately oras part of a fusion protein), each fragment optionally contains one ormore histidine triad motif(s) and/or coiled coil regions of suchpolypeptides. A histidine triad motif is the portion of polypeptide thathas the sequence HxxHxH where H is histidine and x is an amino acidother than histidine. A coiled coil region is a region predicted by“Coils” algorithm Lupus, A et al (1991) Science 252; 1162-1164. In anembodiment the or each fragment includes one or more histidine triadmotif as well as at least one coiled coil region. In an embodiment, theor each fragment contains exactly or at least 2, 3, 4 or 5 histidinetriad motifs (optionally, with native Pht sequence between the 2 or moretriads, or intra-triad sequence that is more than 50, 60, 70, 80, 90 or100% identical to a native pneumococcal intra-triad Pht sequence—e.g.the intra-triad sequence shown in SEQ ID NO: 4 of WO 00/37105 for PhtD).In an embodiment, the or each fragment contains exactly or at least 2, 3or 4 coiled coil regions. In an embodiment a Pht protein disclosedherein includes the full length protein with the signal sequenceattached, the mature full length protein with the signal peptide (forexample 20 amino acids at N-terminus) removed, naturally occurringvariants of Pht protein and immunogenic fragments of Pht protein (e.g.fragments as described above or polypeptides comprising at least 15, 20,30, 40, 50, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500contiguous amino acids from an amino acid sequence in WO00/37105 (SEQ IDNOs 4, 6, 8 or 10) or WO00/39299 (SEQ ID NOs 2, 4, 6, 8, 10 or 14)wherein said polypeptide is capable of eliciting an immune responsespecific for said amino acid sequence in WO00/37105 or WO00/39299. In anembodiment, the PhtX protein is a fragment described in WO 09/12588, forexample those comprising or consisting of the sequences of SEQ ID NO: 2,3 or 4.

In particular, the term “PhtD” as used herein includes the full lengthprotein with the signal sequence attached, the mature full lengthprotein with the signal peptide (for example 20 amino acids atN-terminus) removed, naturally occurring variants of PhtD andimmunogenic fragments of PhtD (e.g. fragments as described above orpolypeptides comprising at least 15 or 20 contiguous amino acids from aPhtD amino acid sequence in WO00/37105 or WO00/39299 wherein saidpolypeptide is capable of eliciting an immune response specific for saidPhtD amino acid sequence in WO00/37105 or WO00/39299 (e.g. SEQ ID NO: 4of WO 00/37105 or SEQ ID NO: 14 of WO 00/39299 for PhtD). All forms ofPhtD mentioned above can be used in the present invention.

In an embodiment of the invention, the method of treatment or preventionis aimed at S. pneumoniae growing in the blood of the patient, forexample for treatment or prevention of septicemia or bacteraemia. In anembodiment, the level of the free concentration of Zn2+ in the blood isless than 10 nM, 7 nM, 5 nM, 3 nM, 2 nM, 1 nM, 700 pM, 500 pM, 300 pM,200 pM 100 pM, 70 pM, 50 pM, 30 pM, 20 pM or 10 pM as measured fromblood serum. The level of Zn2+ may be measured by preparing a serumsample from a blood sample using standard procedures and analysing thesample using graphite furnace absorbance spectrophotometer (GF-AAS) orby using atomic absorption spectroscopy for example by using a VistaAX-CCD simultaneous ICP-AES spectrometer.

In an embodiment of the invention, the bound and free concentration ofZn2+ in the blood is less than 5, 3, 2, 1, 0.5, 0.3, 0.2 or 0.1 mg/l orless than 20, 18, 15, 12, 10, 8, 5, 3, 2, 1, 0.5 or 0.1 μM as measuredfrom blood serum. The level of Zn2+ may be measured by preparing a serumsample from a blood sample using standard procedures and analysing thesample using graphite furnace absorbance spectrophotometer (GF-AAS) orby using atomic absorption spectroscopy for example by using a VistaAX-CCD simultaneous ICP-AES spectrometer.

In an embodiment of the invention, the free concentration of Mn2+ in theblood is less than 10 nM, 7 nM, 5 nM, 3 nM, 2 nM, 1 nM, 700 pM, 500 pM,300 pM, 200 pM 100 pM, 70 pM, 50 pM, 30 pM, 20 pM or 10 pM as measuredfrom blood serum. The level of Mn2+ may be measured by preparing a serumsample from a blood sample using standard procedures and analysing thesample using atomic absorption spectroscopy for example by using a VistaAX-CCD simultaneous ICP-AES spectrometer.

In an embodiment of the invention, the bound and free concentration ofMn2+ in the blood is less than 5, 3, 2, 1, 0.5, 0.3, 0.2 or 0.1 mg/l orless than 20, 18, 15, 12, 10, 8, 5, 3, 2, 1, 0.5, 0.2 or 0.1 μM asmeasured from blood serum. The level of Mn2+ may be measured bypreparing a serum sample from a blood sample using standard proceduresand analysing the sample using atomic absorption spectroscopy forexample by using a Vista AX-CCD simultaneous ICP-AES spectrometer.

In an embodiment of the invention, the method of treatment or preventionis aimed at S. pneumoniae growing in the lung of the patient, forexample the treatment or prevention of pneumonia. In an embodiment, thefree concentration of Zn2+ in the lung is less than 300, 200, 100, 80,50, 20, 10, 5, 3 or 1 μg/kg as measured from a bronchial lavage. In anembodiment, the free concentration of Mn2+ in the lung is less than 300,200, 100, 80, 50, 20, 10, 5, 3 or 1 μg/kg as measured from a bronchiallavage. Optionally, the level of Zn2+ or Mn2+ is measured from a tissuesample in which case the concentration of Zn2+ (or Mn2+) in the lungtissue is less than 20, 15, 10, 5, 2 or 1 μg/g or 300, 200, 150, 100,50, 20, 10, 5, 2, 1, 0.5 or 0.1 μM as measured from lung tissue.Similarly, the level of ions in the tissue sample can be measured byatomic absorption spectroscopy for example by using a Vista AX-CCDsimultaneous ICP-AES spectrometer.

In an embodiment, the S. pneumoniae infection occurs in the acompartment of the ear, for example the middle ear, for example as anotitis media infection. In an embodiment, the level of Zn2+ and/or Mn2+in the middle ear is less than 300, 200, 150, 100, 50, 20, 10, 5, 2, 1,0.5, 0.2 or 0.1 μM.

In an embodiment, the Streptococcus pneumoniae infection occurs in themeninges, for example as a meningitis infection. In an embodiment theconcentration of Zn2+ in the cerebrospinal fluid is less than 1.5, 1,0.75, 0.5, 0.25 or 0.1 μM or less than 100, 75, 50, 40, 25, or 10 μg/L.In an embodiment, the concentration of Mn2+ in the cerebrospinal fluidis less than 2.5, 2, 1.5, 1 or 0.5 μg/L or less than 50, 25, 10 or 5 nM.

In an embodiment of the invention, the human patient has decreasedlevel(s) of Zn2+ and/or Mn2+ as measured by broncheal lavage and/orblood test.

By “decreased level(s)” it is meant that the level of Zn2+ and/or Mn2+as measured by broncheal lavage or blood test is less than that of anaverage human.

In an embodiment of the invention, the human patient to be treated withPhtX is Zn2+ and/or Mn2+ deficient. That is, the level of Zn2+ and/orMn2+ is less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% of theusual level for that body fluid, for example, blood serum, cerebrospinalfluid, interstitial fluid, bronchial lavage.

In an embodiment, the human patient is stressed. The stressed patienthas lower levels of Zn2+ and/or Mn2+ in the body, for example in theblood, interstitial fluid, cerebrospinal fluid and/or lymph.

In an embodiment, the human patient has lower Zn2+ and/or Mn2+ levels inthe body due to previous infection with a bacterial strain, for examplea S. pneumoniae, N. meningitidis, H. influenzae, S. aureus, S.epidermidis, C. difficile, Group A streptococcus, Group B streptococcusand/or M. catarrhalis strain. The previous infection is optionally achronic bacterial infection.

In an embodiment, the administration of PhtX, for example PhtD, is forthe treatment or prevention of Streptococcus pneumoniae infection in theform of scepticaemia, bacteraemia, meningitis, otitis media orpneumonia.

The PhtX protein may also be beneficially combined with further antigensin the method or use of the invention. By combined, it is meant that theimmunogenic composition comprises all of the proteins from within thefollowing combinations, either as carrier proteins or as free proteinsor a mixture of the two. For example, in a combination of two proteinsas set out hereinafter, both proteins may be used as carrier proteins,or both proteins may be present as free proteins, or both may be presentas carrier and as free protein, or one may be present as a carrierprotein and a free protein whilst the other is present only as a carrierprotein or only as a free protein, or one may be present as a carrierprotein and the other as a free protein. Where a combination of threeproteins is given, similar possibilities exist. Combinations include,but are not limited to, PhtD+NR1xR2, PhtD+NR1xR2-Sp91Cterm chimeric orfusion proteins, PhtD+Ply, PhtD+Sp128, PhtD+PsaA, PhtD+PspA,PhtA+NR1xR2, PhtA+NR1xR2-Sp91Cterm chimeric or fusion proteins,PhtA+Ply, PhtA+Sp128, PhtA+PsaA, PhtA+PspA, R1xR2+ PhtD, R1xR2+ PhtA.Optionally, NR1xR2 (or R1xR2) is from CbpA or PspC. Optionally it isfrom CbpA. Other combinations include 3 protein combinations such asPhtD+NR1xR2+ Ply, and PhtA+NR1xR2+ PhtD. In one embodiment, the vaccinecomposition comprises detoxified pneumolysin and PhtD or PhtDE ascarrier proteins. In a further embodiment, the vaccine compositioncomprises detoxified pneumolysin and PhtD or PhtDE as free proteins. Inan embodiment, the combination of proteins comprises PhtD andpneumolysin or PhtD and detoxified pneumolysin. In an embodiment, themethod or use of the invention uses a combination of PhtD, detoxifiedpneumolysin and at least one S. pneumoniae capsulat saccharide,preferably conjugated to a carrier protein.

An aspect, the present invention provides an immunogenic compositioncomprising a PhtX protein and at least 4, 5, 6, 7, 8, 9, 10, 12, 14, 15,16, 17, 18 or 20 S. pneumoniae capsular saccharide conjugates containingsaccharides from different S. pneumoniae serotypes. In such anembodiment, at least one saccharide is conjugated to a PhtX protein suchas PhtD or fusion protein thereof and the immunogenic composition iscapable of eliciting an effective immune response against PhtX, forexample PhtD. In a further aspect of the invention, the immunogeniccomposition comprises at least 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 17,18 or 20 S. pneumoniae capsular saccharide conjugates containingsaccharides from different S. pneumoniae serotypes and a PhtX protein,for example PhtD as a free or unconjugated protein.

In an embodiment, the immunogenic composition of the invention comprisespneumolysin. The pneumolysin is preferably detoxified, for example bychemical treatment or by mutation of at least one amino acid.

The present invention further provides an immunogenic compositioncontaining a pharmaceutically acceptable excipient and/or an adjuvant.

The immunogenic compositions of the present invention may be adjuvanted,particularly when intended for use in an elderly population but also foruse in infant populations. Suitable adjuvants include an aluminum saltsuch as aluminum hydroxide gel or aluminum phosphate or alum, but mayalso be other metal salts such as those of calcium, magnesium, iron orzinc.

The adjuvant is optionally selected to be a preferential inducer of aTH1 type of response. Such high levels of Th1-type cytokines tend tofavour the induction of cell mediated immune responses to a givenantigen, whilst high levels of Th2-type cytokines tend to favour theinduction of humoral immune responses to the antigen.

The distinction of Th1 and Th2-type immune response is not absolute. Inreality an individual will support an immune response which is describedas being predominantly Th1 or predominantly Th2. However, it is oftenconvenient to consider the families of cytokines in terms of thatdescribed in murine CD4 +ve T cell clones by Mosmann and Coffman(Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. (Annual Review of Immunology, 7, p 145-173). Traditionally,Th1-type responses are associated with the production of the INF-γ andIL-2 cytokines by T-lymphocytes. Other cytokines often directlyassociated with the induction of Th1-type immune responses are notproduced by T-cells, such as IL-12. In contrast, Th2-type responses areassociated with the secretion of 11-4, IL-5, IL-6, IL-10. Suitableadjuvant systems which promote a predominantly Th1 response include:Monophosphoryl lipid A or a derivative thereof (or detoxified lipid A ingeneral—see for instance WO2005107798), particularly 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL) (for its preparation see GB 2220211 A);and a combination of monophosphoryl lipid A, optionally 3-de-O-acylatedmonophosphoryl lipid A, together with either an aluminum salt (forinstance aluminum phosphate or aluminum hydroxide) or an oil-in-wateremulsion. In such combinations, antigen and 3D-MPL are contained in thesame particulate structures, allowing for more efficient delivery ofantigenic and immunostimulatory signals. Studies have shown that 3D-MPLis able to further enhance the immunogenicity of an alum-adsorbedantigen [Thoelen et al. Vaccine (1998) 16:708-14; EP 689454-B1].

An enhanced system involves the combination of a monophosphoryl lipid Aand a saponin derivative, particularly the combination of QS21 and3D-MPL as disclosed in WO 94/00153, or a less reactogenic compositionwhere the QS21 is quenched with cholesterol as disclosed in WO 96/33739.A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210. Inone embodiment the immunogenic composition additionally comprises asaponin, which may be QS21. The formulation may also comprise an oil inwater emulsion and tocopherol (WO 95/17210). Unmethylated CpG containingoligonucleotides (WO 96/02555) and other immunomodulatoryoligonucleotides (WO0226757 and WO03507822) are also preferentialinducers of a TH1 response and are suitable for use in the presentinvention.

Oil in water emulsion adjuvants per se have been suggested to be usefulas adjuvant compositions (EP 0 399 843B), also combinations of oil inwater emulsions and other active agents have been described as adjuvantsfor vaccines (WO 95/17210; WO 98/56414; WO 99/12565; WO 99/11241). Otheroil emulsion adjuvants have been described, such as water in oilemulsions (U.S. Pat. No. 5,422,109; EP 0 480 982 B2) and water in oil inwater emulsions (U.S. Pat. No. 5,424,067; EP 0 480 981 B). All of whichform oil emulsion systems (in particular when incorporating tocols) toform adjuvants and compositions of the present invention.

In an embodiment, the oil emulsion (for instance oil in water emulsions)further comprises an emulsifier such as TWEEN 80 and/or a sterol such ascholesterol. In an embodiment, the oil emulsion (optionally oil-in-wateremulsion) comprises a metabolisible, non-toxic oil, such as squalane,squalene or a tocopherol such as alpha tocopherol (and optionally bothsqualene and alpha tocopherol) and optionally an emulsifier (orsurfactant) such as Tween 80. A sterol (e.g. cholesterol) may also beincluded.

The method of producing oil in water emulsions is well known to the manskilled in the art. Commonly, the method comprises mixing thetocol-containing oil phase with a surfactant such as a PBS/TWEEN80™solution, followed by homogenisation using a homogenizer, it would beclear to a man skilled in the art that a method comprising passing themixture twice through a syringe needle would be suitable forhomogenising small volumes of liquid. Equally, the emulsificationprocess in microfluidiser (M110S Microfluidics machine, maximum of 50passes, for a period of 2 minutes at maximum pressure input of 6 bar(output pressure of about 850 bar)) could be adapted by the man skilledin the art to produce smaller or larger volumes of emulsion. Theadaptation could be achieved by routine experimentation comprising themeasurement of the resultant emulsion until a preparation was achievedwith oil droplets of the required diameter.

In an oil in water emulsion, the oil and emulsifier should be in anaqueous carrier. The aqueous carrier may be, for example, phosphatebuffered saline.

The size of the oil droplets found within the stable oil in wateremulsion are optionally less than 1 micron, may be in the range ofsubstantially 30-600 nm, optionally substantially around 30-500 nm indiameter, and optionally substantially 150-500 nm in diameter, and inparticular about 150 nm in diameter as measured by photon correlationspectroscopy. In this regard, 80% of the oil droplets by number shouldbe within the ranges, optionally more than 90% and optionally more than95% of the oil droplets by number are within the defined size ranges.The amounts of the components present in the oil emulsions of thepresent invention are conventionally in the range of from 0.5-20% or 2to 10% oil (of the total dose volume), such as squalene; and whenpresent, from 2 to 10% alpha tocopherol; and from 0.3 to 3% surfactant,such as polyoxyethylene sorbitan monooleate. Optionally the ratio of oil(e.g. squalene): tocol (e.g. α-tocopherol) is equal or less than 1 asthis provides a more stable emulsion. An emulsifier, such as Tween80 orSpan 85 may also be present at a level of about 1%. In some cases it maybe advantageous that the vaccines of the present invention will furthercontain a stabiliser.

Examples of emulsion systems are described in WO 95/17210, WO 99/11241and WO 99/12565 which disclose emulsion adjuvants based on squalene,α-tocopherol, and TWEEN 80, optionally formulated with theimmunostimulants QS21 and/or 3D-MPL.

Thus in an embodiment of the present invention, the adjuvant of theinvention may additionally comprise further immunostimulants, such asLPS or derivatives thereof, and/or saponins. Examples of furtherimmunostimulants are described herein and in “Vaccine Design—The Subunitand Adjuvant Approach” 1995, Pharmaceutical Biotechnology, Volume 6,Eds. Powell, M. F., and Newman, M. J., Plenum Press, New York andLondon, ISBN 0-306-44867-X.

The vaccine preparations containing immunogenic compositions of thepresent invention may be used to protect or treat a mammal susceptibleto infection, by means of administering said vaccine via systemic ormucosal route. These administrations may include injection via theintramuscular (IM), intraperitoneal (IP), intradermal (ID) orsubcutaneous (SC) routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts. Intranasal (IN)administration of vaccines for the treatment of pneumonia or otitismedia is possible (as nasopharyngeal carriage of pneumococci can be moreeffectively prevented, thus attenuating infection at its earlieststage). Although the vaccine of the invention may be administered as asingle dose, components thereof may also be co-administered together atthe same time or at different times (for instance pneumococcalsaccharide conjugates could be administered separately, at the same timeor 1-2 weeks after the administration of the any bacterial proteincomponent of the vaccine for optimal coordination of the immuneresponses with respect to each other). For co-administration, theoptional Th1 adjuvant may be present in any or all of the differentadministrations. In addition to a single route of administration, 2different routes of administration may be used. For example, saccharidesor saccharide conjugates may be administered IM (or ID) and bacterialproteins may be administered IN (or ID). In addition, the vaccines ofthe invention may be administered IM for priming doses and IN forbooster doses.

The content of protein antigens in the vaccine will typically be in therange 1-100 μg, optionally 5-50 μg, e.g. in the range 5-25 μg. Followingan initial vaccination, subjects may receive one or several boosterimmunizations adequately spaced.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

The vaccines or immunogenic compositions of the present invention may bestored in solution or lyophilized. In an embodiment, the solution islyophilized in the presence of a sugar acting as an amorphouslyoprotectant, such as sucrose, trehalose, glucose, mannose, maltose orlactose. In an embodiment, the solution is lyophilized in the presenceof a sugar acting as an amorphous lyoprotectant, and a bulking agentproviding improved cake structure such as glycine or mannitol. Thepresence of a crystalline bulking agent allows for shorteningfreeze-drying cycles, in the presence of high salt concentration.Examples of such mixtures for use in lyophilisation of the immunogeniccompositions or vaccines of the invention include sucrose/glycine,trehalose/glycine, glucose/glycine, mannose/glycine, maltose/glycine,sucrose/mannitol/trehalose/mannitol, glucose/mannitol, mannose/mannitoland maltose/mannitol. Typically The molar ratio of the two constituentsis optionally 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6. Immunogenic compositionsof the invention optionally comprise the lyophilisation reagentsdescribed above.

The above stabilising agents and mixtures of stabilising agents canfurther include a polymer capable of increasing the glass transitiontemperature (Tg′) of the formulation, such as poly(vinyl-pyrrolidone)(PVP), hydroxyethyl starch or dextran, or a polymer acting as acrystalline bulking agent such as polyethylene glycol (PEG) for examplehaving a molecular weight between 1500 and 6000 and dextran.

Although the immunogenic compositions of the present invention may beadministered by any route, administration of the described vaccines intothe skin (ID) forms one embodiment of the present invention. Human skincomprises an outer “horny” cuticle, called the stratum corneum, whichoverlays the epidermis. Underneath this epidermis is a layer called thedermis, which in turn overlays the subcutaneous tissue. Researchers haveshown that injection of a vaccine into the skin, and in particular thedermis, stimulates an immune response, which may also be associated witha number of additional advantages. Intradermal vaccination with thevaccines described herein forms an optional feature of the presentinvention.

The conventional technique of intradermal injection, the “mantouxprocedure”, comprises steps of cleaning the skin, and then stretchingwith one hand, and with the bevel of a narrow gauge needle (26-31 gauge)facing upwards the needle is inserted at an angle of between 10-15°.Once the bevel of the needle is inserted, the barrel of the needle islowered and further advanced whilst providing a slight pressure toelevate it under the skin. The liquid is then injected very slowlythereby forming a bleb or bump on the skin surface, followed by slowwithdrawal of the needle.

More recently, devices that are specifically designed to administerliquid agents into or across the skin have been described, for examplethe devices described in WO 99/34850 and EP 1092444, also the jetinjection devices described for example in WO 01/13977; U.S. Pat. No.5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat.No. 5,993,412, U.S. Pat. No. 5,649,912, U.S. Pat. No. 5,569,189, U.S.Pat. No. 5,704,911, U.S. Pat. No. 5,383,851, U.S. Pat. No. 5,893,397,U.S. Pat. No. 5,466,220, U.S. Pat. No. 5,339,163, U.S. Pat. No.5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Pat.No. 5,520,639, U.S. Pat. No. 4,596,556, U.S. Pat. No. 4,790,824, U.S.Pat. No. 4,941,880, U.S. Pat. No. 4,940,460, WO 97/37705 and WO97/13537. Alternative methods of intradermal administration of thevaccine preparations may include conventional syringes and needles, ordevices designed for ballistic delivery of solid vaccines (WO 99/27961),or transdermal patches (WO 97/48440; WO 98/28037); or applied to thesurface of the skin (transdermal or transcutaneous delivery WO 98/20734;WO 98/28037).

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance.

Embodiments herein relating to “vaccine compositions” of the inventionare also applicable to embodiments relating to “immunogeniccompositions” of the invention, and vice versa.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES Methods Animals

OF1 and CBA/J female mice used in this study were purchased from CharlesRiver laboratories (Lyon, France). Balb/c mice were from Harlan (Horst,The Netherlands). All experiments and assays were performed atGlaxoSmithKline Biologicals (GSK, Rixensart, Belgium) in accordance withthe Belgian national guidelines for animal experimentation.

Bacterial Strains and Culture Conditions.

The strain 2/D39 was kindly provided by JC Paton (University ofAdelaide, Australia). The strains 4/CDC and 6B/CDC were obtained fromthe Center for Disease Control and Prevention (CDC), and the 19F/2737strain from the American type culture collection (ATCC). The strain 3/43was provided by E Yourassowski (Brugmann Hospital, University ofBrussel, Belgium).

S. pneumoniae strain TIGR4 {Tettelin 2001} was kindly provided by AndrewCamilli (Tufts University School of Medicine, Boston, Mass., USA). TheWU2 strain was kindly provided by David E Briles (University of Alabamaat Birmingham, Birmingham, Ala., USA). The type 4 strain was obtainedfrom the CDC (Center for Disease Control & Prevention).

Pneumococci were routinely grown in Todd-Hewitt broth (THB, Difco) with0.5% (w/v) yeast extract at 37° C./8% CO₂. When appropriate,erythromycin and/or spectinomycin (Sigma-Aldrich, Bornem, Belgium) wasadded at a concentration of 0.2 and 250 μg/ml; respectively.

Escherichia coli DH5α and JM109 strains (Gibco BRL, Life Technology)were grown in Luria-Bertani broth (LBT, Difco) with or without 1.5%(w/v) Bacto-agar (Difco) at 37° C. for 16 h. When appropriate,erythromycin or spectinomycin was added to the growth medium at aconcentration of 100 μg/ml.

For the study on Pht occurrence, besides 23 in-house and pneumococcalmolecular epidemiology network (PMEN) strains, 34 isolates were providedby TJ Mitchell (Scotland), 6 by RE Gertz (USA), 2 by AB Brueggemann (UK)and 9 were from the American Type Culture Collection (ATCC).

Antigens

CbpA (or PspC) was a truncated recombinant protein, as described inBrookes-Walter et al J. Infect. Dis. 67; 6533-6542 (1999), kindlyprovided by JC Paton. The protein was constructed from the sequence ofthe D39 strain and belongs thus to clade A. PspA (clade 2) and PsaA arerecombinant proteins originating from the 2/D39 strain Ogunniyi et alInfect. Immun. 68; 3028-3033 (2000), both provided by JC Paton.

DNA Treatment and Analysis.

Escherichia coli plasmid DNA was obtained using Plasmid Midi or MiniPurification Kit (Qiagen Benelux, Venlo, The Netherlands). PCR productswere purified with the QIAquick PCR Purification Kit and DNA digestswere purified on 1% (w/v) agarose gel using the QIAquick Gel ExtractionKit (Qiagen). Restriction and ligation enzymes were obtained from NewEngland BioLabs (Westburg, Leusden, Belgium). The Expand High FidelitySystem (Roche, Mannheim, Germany) was used for each PCR reaction ofthese studies. All commercial products were used under conditionsrecommended by the suppliers.

DNA sequencing was carried out with the Big Dye Terminator SequencingKit on an Applied Biosystems automated DNA sequencer (model 3100)(Applied Biosystems Inc, Forster City, Calif., USA). Sequence analyzeswere performed with the MacVector V6.5 software (Oxford Molecular Ltd.,Madison) or the Vector NTI 7.1 software (Informax), and sequencescompared to the available S. pneumoniae TIGR4 genome sequence(www.tigr.org) {Peterson 2001}.

S. pneumoniae Genomic DNA Extraction.

Chromosomal DNA from each strain was obtained by harvesting confluentovernight growth from one or two heavily inoculated blood agar platesinto 1 ml of TE (10 mM Tris-HCl; 5 mM EDTA; pH 7.8). The bacterialsuspension was centrifuged for 5 minutes at maximal speed in amicrocentrifuge and the pellet was resuspended in 75 μl of TE. Celllysates were obtained by sequential addition of 20 μl of lysozyme (100mg/ml) and 20 μl of proteinase K (20 mg/ml) and incubation at 37° C., 45minutes. Then, 500 μl of lysis buffer (10 mM Tris-HCl, pH 8.0; 0.14 MNaCl; 0.1 M sodium citrate; 1 mM EDTA, pH 8.0; 0.1% (w/v) sodiumdeoxycholate) was added and incubated for 10 minutes at roomtemperature. At the end of this incubation period, 250 μl of ammoniumacetate (7.5 mM, pH 7.7) was added to crude lysate and incubated 10minutes on ice. The viscous DNA was extracted twice withphenol/chloroform/isoamyl (25:24:1) and precipitated in isopropylalcohol. The resulting DNA was washed with 70% (v/v) ethanol andresuspended in 50 μl TE containing 0.6 μl RNaseA (10 mg/ml). DNAsuspensions were stored at 4° C.

RNA Isolation.

Total RNA was isolated from pneumococci grown from an optical density at600 nm (OD₆₀₀) of 0.01 in THY to different OD₆₀₀ to evaluate geneexpression at different growth phases (early log, OD₆₀₀=0.3; late log,OD₆₀₀=0.9; stationary, OD₆₀₀=1.2). Cells were centrifuged andresuspended in RNase-free Tris-EDTA containing 6 mg lysozyme ml⁻¹ and 1mg sodium deoxycholate ml⁻¹, and incubated at room temperature for 10min. After incubation, RNA isolation was performed with the QIAGENRNeasy Mini Kit following manufacturer's instructions. Contaminatinggenomic DNA was eliminated by incubating RNA samples with 1 unit ofDNase I per μg of RNA for 1 h at 37° C., followed by DNase inactivationwith 2.5 mM EDTA for 10 min at 65° C. Total RNA was quantified using theRibogreen® RNA Quantification Kit (Molecular Probes) followingmanufacturer's instructions.

5′-Rapid Amplification of cDNA End (RACE).

The method used to identify transcription starts was adapted fromRanasinghe & Hobbs {Ranasinghe 1998}. Briefly, a primer specific for the3′ end of the phtE gene was used to synthesize the first-strandcomplementary DNA (cDNA) from total RNA with the Superscript II reversetranscriptase (Invitrogen), following manufacturer's instructions. RNaseA was then added for 1 h at room temperature to generate blunt 3′ endson the cDNA-RNA hybrid. The hybrid was inserted into EcoRV-digested pKSplasmid (Stratagene) using T4 DNA ligase (incubation overnight, 16° C.).A PCR reaction was set up to amplify the 5′ end using another reverse 3′end-specific phtE primer and pKS-specific T7 promoter primer. Sequencingof the pKS-cDNA junction was performed to identify the +1 base.

Transcriptional Terminator Identification.

Terminator identification was performed using the Wisconsin SequenceAnalysis Package version 10.1 (Genetics Computer Group) based on themethod of Brendel & Trifonov {Brendel 1984}.

RT-PCR.

RT-PCR studies were performed as follows. RNA (2 μg) was first denaturedfor 5 min at 65° C. in a mixture containing 10 μM of 3′-endgene-specific reverse primer and 20 units of RNaseOut in a total volumeof 10 μl. The reverse transcription reaction was then carried out byadding 5 mM dithiothreitol, 1 mM dNTP, 15 units of Thermoscript reversetranscriptase (Invitrogen), 1×cDNA synthesis buffer and RNase-freesterile water to a volume of 20 μl. The reverse transcription mixturewas incubated at 56-58° C. for 1 hour, followed by reverse transcriptasedenaturation for 5 min at 85° C. The RNA strand on the RNA-cDNA hybridswas degraded by incubating the reverse transcription solution at 37° C.for 20 min with 1 unit of RNase H. The PCR reaction was carried with 2μl cDNA using different 5′ gene-specific forward primers and the 3′gene-specific reverse primers used for the reverse transcriptionreaction (0.5 μM final concentrations), 0.2 mM dNTP, Taq DNA polymerasereaction buffer, 2.5 units of Taq DNA polymerase (Amersham Biosciences)and sterile water to a volume of 50 μl. The PCR cycle consisted ofinitial denaturation at 94° C. for 5 min, followed by 25-30 cycles ofdenaturation at 94° C. for 15-30 sec, annealing at 55° C. (phtE, phtD)or 63° C. (phtB, D, A) for 15-30 sec and extension at 72° C. for 1 min,and completed by a final extension step at 72° C. for 5-7 min. Anegative control composed of RNA without reverse transcription reactionwas also conducted to exclude DNA contamination in the RNA preparation.PCR products were separated by 1% (w/v) agarose gel electrophoresis andvisualized by ethidium bromide staining.

Preparation of Pht Mutants.

Mutator vectors were constructed from the pGEM-T vector (PromegaBenelux, Leiden, the Netherlands) that replicates in E. coli but not inS. pneumoniae. They contain recombinant zones that correspond to theupstream and downstream regions of the pht genes to be deleted,amplified by PCR, surrounding an antibiotic-resistance gene (primers andrestriction sites used to contruct the mutator vectors can be given onrequest). To prepare the quadruple Pht-deficient mutant, two differentantibiotic resistance genes had to be used in order to combine deletionin the two different loci (locus phtD/phtE, and locus phtA/phtB). Anerythromycin resistance gene (ermB), amplified from a derivative of thepJDC9 vector, was selected for the phtD/phtE locus. For the phtA/phtBlocus, a spectinomycin resistance gene (aad(9) gene), purified from thepR350 plasmid (kindly provided by J Paton) was used.

Cloning was performed in DH5α or JM109 E. coli strains, with thedifferent constructed plasmids and plated on LB agar with the respectiveantibiotics. Transformation of E. coli with plasmid DNA was carried outby standard methods with CaCl₂-treated cells {Hanahan 1985}.

The 4/CDC S. pneumoniae strain was prepared for transformation by twosuccessive growing steps, before resuspension in CTM medium (10 g/lCasamino acids; 5 g/l tryptone; 5 g/l NaCL; 10 g/l yeast extract; 0.4 MK₂HPO₄; 20% glucose; 30 mg/ml glutamine; 1% BSA, 0.1 M CaCL₂; pH 7.8)aliquoting, and freezing in 15% glycerol. Those aliquots were used fortransformation. After thawing, CSP-1 or CSP-2 (100 ng/ml in CTM medium)was added to induce competence, and the bacteria were incubated at 37°C. Different time points were taken (5, 10, 15, and 20 min) to optimisecompetence. After addition of 1 μg of mutator vector, cells wereincubated at 32° C. for 30 min, with shaking, followed by 2-4 h at 37°C., under 5% CO₂. At last, bacteria were plated on blood agar with theappropriate antibiotics. For the quadruple mutant, the PhtD,E-KO strainwas transformed with the plasmid that brings PhtA,B deficiency by usingthe same protocol as described above.

SDS-PAGE and Western Blot Analysis.

Heat-killed bacterial suspensions were obtained by harvesting theconfluent overnight growth from 5 heavily inoculated blood agar platesinto 1 ml of sterile PBS (0.14 M NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mMKH₂PO₄, pH 7.2), and an incubation step at 56° C. for 45 minutes. Then,sample buffer (60 mM Trizma base, 1% (w/v) SDS, 10% (v/v) glycerol,0.01% (w/v) bromophenol blue, 2% (v/v) β-mercaptoethanol) was added tothe heat-killed suspensions. Preparations were boiled for 5 minutes,centrifuged at maximum speed in a microcentrifuge for 2 minutes andseparated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) asdescribed by Laemmli {Laemmli 1970}. Proteins were electrophoreticallytransferred from acrylamide gels onto nitrocellulose membranes (Bio-Rad,Richmond, Calif.), as described {Towbin 1979}. Membranes were probedwith a mouse polyclonal antibody raised against PhtD, followed by goatanti-mouse IgG conjugated to alkaline phosphatase (Promega Benelux.).Enzyme-labelled bands were visualized with a NBT/BCIP substrate system.

Culture Growth in Ion-Deficient Medium.

Wild-type 4/CDC strain, and corresponding PhtD- and Phtquadruple-deficient mutants were cultured under different conditions ofion depletion or supplementation in a chemically defined syntheticmedium (MS){SICARD 1964}. MS medium was supplemented by increasingconcentrations of, alternatively, Mn²⁺, Fe²⁺, Fe³⁺, Cu²⁺, or Zn²⁺.Optical density at 600 nm was monitored during log-phase and atstationary phase. Results were compared with those of wild-type.

Wild-type WU2 strain was cultured with or without the Zn-specificchelator N,N,N′,N′,-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) toobserve the effect of zinc depletion on Pht expression at the RNA (byRT-PCR) and protein (by flow cytometry) levels.

Flow Cytometry

WU2 bacteria were grown in THB+0.5% yeast extract at 37° C., 8% CO₂, upto log-phase. Alternatively, TPEN 30 μM, a zinc chelator, was added tothe medium. After centrifugation, bacterial pellets were resuspended ina solution containing anti-PhtE, anti-PhtB/D, anti PhtD/E or anti-type 3polysaccharide monoclonal antibodies as control. After 2 h at 4° C.; thesolutions were centrifuged, the bacterial pellets were washed in PBS-BSA2% before they were incubated for 1 h at room temperature in AlexaFluor™(Molecular Probes)-conjugated goat anti-mouse secondary antibody inPBS-BSA 2%. After washing, cells were fixed in PBS-formaldehyde 0.25%and FACS analysis was performed. The median of surface fluorescence wasrecorded.

Determination of Pht Occurrence

In order to select representative strains of S. pneumoniae, thepopulation structure was analysed according to the strain genotype asdetermined by MLST (Multi-Locus Sequence Type; www.mlst.net). Based onMLST isolate Sequence Type (ST), major clonal lineages were determined.For each group, a representative strain was selected for occurrenceanalysis, which was carried out by Western blotting on whole bacterialextracts with anti-PhtD polyclonal antibodies (cross-reactive with A, Band D) or anti-PhtE, and by PCR on pneumococcal genomic DNA usingprimers specific for PhtA, B, D or E.

DNA Sequencing for PhtD Conservation Analysis

DNA of 107 MLST-selected strains was PCR-amplified using PhtD-specificoligonucleotide primers. The 107 sequences were aligned by ClustaIXprogram and the identity was calculated by the Superneedle program(percentage of identity is 100×(number of identities/length of shortestsequence).

Example 1 Characterization of the pht Genes Genomic Organization of thepht Genes.

In a previous work, DNA sequencing of overlapping clones from a S.pneumoniae strain SP64 genomic library {Hamel 2004} and PCR analysesallowed deduction of the genomic organization of the pht genes and theirneighbouring genes in this type 6B strain. The phtA and phtB genes, aswell as the PhtD and the PhtE genes, were organized as a pair. BLASTanalyses on the TIGR web site (www.tigr.org) {Peterson 2001} indicatedthat the two gene tandems were located about 161 kbp apart in the S.pneumoniae TIGR4 genome and that the genomic organization was identicalto the one observed in the SP64 strain (FIG. 1). The same pht geneorganization was also found in the 4/CDC strain and in the WU2 strain,with the exception that phtA is missing in the latter (data not shown).Sequencing of the pht genes surroundings on the TIGR4 strain DNAconfirmed the latter observation (data not shown). Additional analysisdemonstrated that phtA and phtB were separated by 157 bp, whereas phtDand phtE were separated by 209 bp in the TIGR4 strain, which was chosenfor further work.

On the phtD-phtE tandem side, a gene presenting 72% sequence similarityto the group A and B streptococci lmb genes, coding for laminin-bindingproteins (accession # AAK34689 {Ferretti 2001} and AAD13796 {Spellerberg1999} respectively), was located 7 bp upstream of the phtD gene (FIG.1). This gene product was also recently denominated as AdcAII, anddescribed as an ABC transporter-like zinc-binding protein {Loisel 2008}.A 1392-bp ORF, located 142 bp upstream of the lmb gene homolog, codesfor a protein presenting 64% sequence similarity with the Bacillussubtilis metabolite transporter YfnA protein (accession # D69814) and81% similarity with a putative amino acid permease of S. pyogenes(accession # AAK33157) {Ferretti 2001, Kunst 1997}. A sequencepresenting 79% identity with the first 481 bp of phtE (proposed phtF)was found 226 bp after the phtE stop codon (FIG. 1). This sequence alsoshows 72% identity with the phtA, B and D genes.

On the phtA-phtB tandem side, a 1332-bp ORF, presenting 73% sequencesimilarity with the Streptococcus salivarius ptsI gene (accession numberP30299) {Gagnon 1992}, was located 253 bp upstream of the phtA gene(FIG. 1). This gene presented a frameshift in the TIGR4 strain that wesequenced compared to the ptsI gene found in the whole genome sequence(accession # AAK75285). No functional ORF was located immediatelydownstream of both gene pairs.

Example 2 Transcriptional Organization of pht Genes

The genomic organization of pht genes suggested that the tandem genesmight be coordinately transcribed. Further studies were thus performedto examine this hypothesis. First, putative promoters and ribosomebinding sites of pht genes were identified. The 5′-RACE on the phtE geneallowed the identification of its transcription start, from which thepromoter region was deduced. The transcription start site (+1) was foundto be located 96 bases upstream of the PhtE translation start site,downstream of typical S. pneumoniae −10 and −35 RNA polymerase bindingsites {Morrison 1990} and upstream of a ribosome binding site (FIG. 2a). Similar sequence organization was found upstream of the phtA, phtBand yfnA genes, indicating the presence of putative promoters (FIG. 2 b,c and e). However, due to the close proximity of the lmb gene (7 bp), nopromoter sequence was identified for the phtD gene. On the other hand, asequence identical to the −35 sequences of the other pht genes waslocated upstream of the lmb gene (FIG. 2 d). Ribosome binding sites wereobserved 5 to 7 bp upstream of all start codons.

Transcription termination sites of pht and adjacent genes were alsoidentified. Computer analysis of predicted mRNA secondary structuressuggested the presence of stem-loop terminator-like structures at the 3′ends of genes. Hairpin structures could form with calculated freeenergies of dissociation (ΔG) of −9.4, −27.0, −16.8, and −21.6 kcalmol⁻¹ for phtB, phtD, phtA, and ptsI respectively, as determined by themethod of Turner et al. (1988) {Turner 1988} (FIG. 3). In fact, theterminator identified for the phtD gene was identical to the onereported by the TIGR web site for ORF SP1003, which corresponds to thephtD gene homolog (www.tigr.org). No transcription terminators wereidentified by the TIGR group for the other pht or surrounding genes,probably reflecting differences in algorithms used by both studies. Mosthairpins ended with a stretch of T residues as typically found inprokaryotic transcription terminators {Rosenberg 1979} and were locatedwithin 70 bp downstream of stop codons (FIG. 3). Interestingly, the phtEterminator sequence (ΔG of −4.7 kcal mol)⁻¹ was located 1867 bpdownstream of its stop codon and of the phtF gene, the latter ORFcontaining in-frame stop codons preventing its translation (FIG. 3 a).No terminator sequences were identified downstream of yfnA and lmbgenes.

The genomic organization suggested that phtE could be part of an operoncomposed of the yfnA, lmb, phtD, phtE and phtF genes. Nevertheless, the5′-RACE (FIG. 2 a) and terminator identification (FIG. 3 a) indicatedthat phtE was the first gene transcribed on a bicistronic message,composed of phtE and phtF genes, which was confirmed by RT-PCR. Theregions phtE to phtF were amplified by RT-PCR (FIG. 4 a, lanes 4 and 6),whereas no amplification product was obtained with primer pair specificto the region between genes phtD and phtE (FIG. 4 a, lane 5), indicatingtranscriptional termination downstream of phtD (FIG. 3 c).

As shown in FIG. 4 a (lanes 1 to 3), RT-PCR amplified the regions yfnAto phtD. Moreover, Loisel et al. {Loisel 2008} have demonstrated thatthis phtD transcript also encodes, in addition to yfnA, lmb and phtD,the two genes upstream yfnA (ccdA that is involved in the biogenesis ofcytochrome c and spr0904 that displays similarity with thioredoxine).Interestingly, the identification of a putative promoter upstream of thelmb gene (FIG. 2 d) suggested transcriptional coupling of the phtD andlmb genes.

Results obtained for phtB and phtA genes showed that they weretranscribed as monocistronic mRNAs, as was suggested by promoter (FIGS.2 b and c) and terminator sites identification (FIGS. 3 b and d).Analysis of the transcriptional organization of phtA and phtB by RT-PCRrevealed a phtB-specific amplicon with phtB-specific primers (FIG. 4 a,lane 7). No amplification product was obtained by RT-PCR with primersamplifying the region between phtA and phtB (FIG. 4 a, lane 8),indicating a monocistronic organization of the phtA and phtB genes.Terminator site identification (FIG. 3 e) indicated that ptsl istranscribed as a monocistronic message, which also confirmed that phtAis not part of a polycistronic transcript.

Example 3 Construction and Use of Pht Mutants Characterization of theMutants.

The mutants PhtA⁻, PhtB⁻, PhtD⁻, PhtE⁻, and the quadruple mutantPhtABDE⁻ were constructed. To assess the accuracy of the recombination,the genomic DNA of the mutant strains was purified and the recombinantregions were sequenced (data not shown). Furthermore, the mutants werephenotypically characterized by immunoblotting, using a mouse polyclonalanti-PhtD antibody (FIG. 5). All four Pht isotypes were recognized bythis antibody. However, PhtE bands were fainter, confirming the greatestdivergence of this Pht from the three others.

The Influence of Various Ions on Bacterial Growth

The growth of the Pht quadruple mutant was dramatically decreased in MSmedium, compared to that of the wild-type strain and of the differentPht single mutants (FIG. 6 a). When the medium was supplemented with upto 200 μM of Fe²⁺, Zn²⁺, or Mn²⁺, the growth of the wild-type and of thePhtD-deficient mutant was slightly ameliorated (growth rate vs MS alone:96-130%). In contrast, the behavior of the quadruple mutant wasstriking. While the addition of 200 μM of Fe²⁺ to MS only induced a25.3% increase of growth (FIG. 6 d), the same concentration of Zn²⁺ orMn²⁺ restored the growth capacity of the quadruple mutant (FIG. 6 b,c).This represents up to 92.3% increase in growth rate, compared to thatobtained in MS alone. However, this recovery of growth rate was delayed,only visible after overnight incubation, as no improvement was visiblewithin the first hours of culture. The addition of Mg²⁺ did not restoregrowth completely at 200 μM, as did Zn²⁺ or Mn²⁺, but similar increasein growth rates were obtained when 1 mg/ml of Mg²⁺ was added to MS (datanot shown). The addition of high concentrations of Cu²⁺ was toxic forwild-type and mutant strains (data not shown).

Example 4 The Effect of Zinc Depletion on pht Expression

When the zinc chelator TPEN was added to the culture medium, theexpression level of the Pht proteins was increased, as determined byflow cytometry experiments (FIG. 7 a,b,c). As a control, no shift inmean fluorescence was observed with anti-type 3 polysaccharide antibodyin the same conditions of zinc depletion (FIG. 7 d). At the RNA level,we could measure, by RT-PCR, up to 25-fold increase in the phtEtranscription level in the condition of zinc depletion (data not shown).

Example 5 Pht Occurrence in Pneumococcus

In total, 74 strains (including 23 PMEN and in-house strains) wereinvestigated. In this set of representative strains, 18 clonal lineageswere represented, 46 strains (61%) with 27 different ST belonged to the3 major clonal groups (1, 11 and 23), 56 different ST were present,among which the more represented were 81, 90, 124, 156, 162 and 199 (22strains), and 27 different serotypes were present among which the morerepresented were 19F, 6B, 3, and 23F (47% of all strains).

By PCR on genomic DNA, we found the genes for PhtD, PhtE, PhtB and PhtAin 100%, 97%, 81%, and 62% of the strains, respectively. Fifty-fourpercent of the strains were found to carry the four pht genes in theirgenome. On immunoblots with polyclonal antibodies raised against PhtD,we could detect PhtD in all strains. Likewise, the other Phts were foundby immunoblotting in all strains that carry their respective genes.Notably, due to the highest genetic divergence, PhtE was better detectedwith a polyclonal antibody specifically raised against it (FIG. 8). Somepeculiar Phts were found, such as a PhtE of a lower size (10-kDasmaller) in 6 isolates, and of an even smaller size (20-kDa less) in 8strains. Likewise, 4 strains were found to produce a truncated PhtA(FIG. 8), which gene was not detected by PCR. Interestingly, these 4strains also expressed the 20-kDa-truncated PhtE. At least, sequencingof the phtA/B locus of phtB negative strains has revealed that the onlygene present in this locus was an hybrid between either phtA and phtB orphtA and phtD genes.

Interestingly, sequence analysis has demonstrated that the signalsequence encoded by pht genes was specific for each Pht family members.Indeed, the specific signal sequence of a Pht family member differ atleast in one position to the signal sequence of another Pht familymember (see table 1).

Next, it was attempted to determine whether links can be made betweenthe Pht expression profile and the isolate genotype/serotype. In thestrains analyzed, all serotype 2, 4, 14, 6B and 7F isolates possessedthe 4 Phts, and all serotype 3, 9, 19F and 22F isolates lacked PhtA orcarried a smaller PhtA.

About a potential link between MLST genotype and Pht expression profile,the following features could be determined: the 10-kDa-truncated PhtEwas found mainly in the genotype ST 199 group. The serotypes of thesestrains are 19F, 19A, 15A, 1 and 6A. The 20-kDa-truncated PhtE wasobserved in 8 isolates that all belonged to the same clonal lineage(group 1), but carrying different serotypes (9, 19A, 19F, 14). At last,strains lacking PhtA were observed in different clonal lineages.Therefore, no major link between lack of PhtA and genotype wasidentified.

Example 6 PhtD Conservation

In the Pht occurrence study, PhtD was found to be present among allpneumococcal strains tested, which designates it as the best vaccinecandidate among the Pht family. In this respect, it was found essentialto determine the level of sequence conservation among pneumococcalstrains. For that, DNA sequencing was carried out.

From the analysis of 107 strains (based on MLST classification), it wasdetermined that the length of PhtD varies between 831 and 853amino-acids with a molecular mass of around 100 kDa. PhtD was found tobe highly conserved among the 107 strains tested and only 1 sequencedisplayed a stop for a truncated protein (strain 4/75). The proline-richregion contained 13-15 prolines for all strains (in 7 strains, only11-13 prolines). Limited stretches of variability <4 amino acids werefound in the sequence of PhtD.

DISCUSSION

The Pht proteins are promising candidates to be incorporated in avaccine against pneumococcal infectious diseases. In that respect, itappeared crucial to investigate how the expression of these proteins isregulated, in order to better define their role in pneumococcalpathogenesis.

Genome analysis showed that the four gene homologues are arranged intandem. The presence of a fifth member, though truncated, of the phtgene family, downstream of the phtE gene was also evidenced, confirmingthe finding in a previous study {Adamou 2001}. It seems that thistruncation is conserved since the same organization was found in the S.pneumoniae strain R6 genome (accession # AAK99714) {Hoskins 2001}.

Our study showed that the tandem organization of the pht genes does notcorrelate with a pht bicistronic transcription. None of these genes wereco-transcribed with their related pht neighbor, under the conditionstested. Promoter and terminator analyses correlated well withtraditional RT-PCR studies. We evidenced that the phtB, phtA and phtEgenes all possess individual putative promoters and that mRNAtranscription probably ends soon after the corresponding stop codons. Onthe other hand, a peculiarity of the phtD gene was observed. Indeed, nopromoter was identified in-silico for phtD. Instead, promoters, but notranscription terminators, were identified for lmb and yfnA genes, twogenes located upstream of phtD, which tended to indicate that thosegenes are organized in an operon system. This corroborates the recentfinding that phtD may be expressed in a large operon system togetherwith the 4 genes upstream {Loisel 2008}. Nevertheless, the fact that apromoter was identified for yfnA and for lmb indicates thattranscription may start at these locations, which means thatphtD-containing transcripts of different length may be produced. Inaddition, an adcR binding site was identified upstream of the lmb gene{Loisel 2008, Panina 2003}, which tend to say that a zinc-regulatedbicistronic transcript with lmb and phtD may also exist, which has beensuggested by other authors. Studies by Spellerberg et al. {Spellerberg1999} showed that the group B streptococcal (GBS) lmb gene isco-transcribed with a gene whose product presents 67% sequencesimilarity with the first 225 (phtE) and first 228 (phtA, phtD and phtB)amino acids of pht gene products (accession # AF062533). A comparablegenomic arrangement was also observed in the group A streptococcal (GAS)genome {Ferretti 2001}. Further, it was proposed that co-transcriptionof lmb and phtD might indicate a functional link, with the latter geneproduct being involved in pneumococcal adhesion and invasion {Panina2003}.

It is interesting to note that the phtD gene can be transcribed as apolycistronic message with those two other genes, yfnA and lmb, that maybe involved in transport and specific binding activities, respectively.Indeed, YfnA in S. pneumoniae {Hoskins 2001}, and the homologousproteins in B. subtilis {Yamamoto 1997}, S. pyogenes {Ferretti 2001},and S. mutans {Ajdic 2002} are thought to be amino-acid transporters,members of the superfamily of permeases. As to the Lmb protein, it hasbeen described as an ABC transporter-like zinc-binding protein {Loisel2008} and a putative laminin-binding protein {Spellerberg 1999}. Indeed,this protein demonstrates similarities with an adhesin family known asLraI, found initially in oral streptococci {Jenkinson 1994}, but sincethen discovered in other streptococci and genera {Cockayne 1998}. It wassuggested that Lral-like proteins are involved in the colonization ofhuman epithelium by streptococci and their subsequent invasion into thebloodstream {Elsner 2002}. It is not clear why yfnA, lmb, and phtD areassociated in an operon system. One plausible hypothesis is that thosethree proteins are required at the same moment of the bacterial cyclus,for invasion or growth, for instance, without necessarily beingassociated in their functions. However, the determination of the role ofthe Pht proteins might give some clue for this genomic association. Wecan speculate that similarities between intra-species Pht proteins areindicative of interchangeable roles. It might also be that the proteinsshare similar functions through their homologous regions and, at thesame time, exert distinct activities, even at different developmentphases of the bacterium. The results we have obtained in immunoblottingwith protein extracts from the various Pht-deficient mutants that wehave produced tend to show that there is no compensation for gene lossby increasing the level of expression of the remaining Pht geneproducts. This feature was also described recently at the RNA level, byusing RT-PCR {Ogunniyi 2009}.

As already mentioned, all Pht proteins share histidine triad motifs{Adamou 2001, Hamel 2004, Zhang 2001}, thought to be involved in metalbinding. Interestingly, it has been speculated that these motifs mightbe involved in zinc binding, especially to generate conformationallyfunctional Pht proteins {Panina 2003}. The same authors alsohypothesized that a zinc-restricted environment could induce theexpression of the Pht proteins and favor Streptococcus colonization andinvasion. In this context, we carried out experiments in which wild-typeand Pht-deficient strains were cultured under different conditions ofion depletion and supplementation. In a minimal synthetic medium,wild-type and PhtD-deficient strains grew more slowly than in rich LBmedium, but almost no growth of the quadruple Pht-deficient mutant wasobserved in the minimal medium. Strikingly, when Zn²⁺ or Mn²⁺ was added,and this was particularly visible at concentrations in the range of20-200 μM, the growth of the quadruple mutant was restored up to that ofthe wild-type. However, our results show that the growth of thequadruple mutant was delayed, as compared with the wild-type.

These observations, besides confirming the requirement of Zn²⁺ and Mn²⁺for bacterial growth, argue for a critical role of the Pht family inZn²⁺ and Mn²⁺ uptake. The fact, as we have observed, that Zn²⁺deprivation induces de novo synthesis of proteins of the Pht family is afurther argument to support a tight relation between Pht and Zn²⁺. Thisregulation is likely to occur through AdcR protein that regulates zincuptake in S. pneumoniae. Indeed, putative binding sites for AdcR proteinhave been found upstream of the phtA, phtB, and phtE genes, and of thelmb-phtD operon {Panina 2003}. Binding of AdcR, induced in conditions ofhigh Zn²⁺ concentrations, inhibits the transcription of the genes underits dependence. Upon direct or indirect zinc starvation conditions,hence reduction in intracellular concentration of this metal, repressionby AdcR is relieved {Brenot 2007, Clayerys 2001}. Conversely to that andto what we have observed in the present study, it was recently publishedthat the addition of zinc in culture medium elicits Pht production{Ogunniyi 2009}. However, the two methods used were distinct in thesense that, in the present work, zinc was removed from a zinc-richmedium while Ogunniyi et al. added zinc to a zinc-poor medium. It isreasonable to estimate that Pht production is regulated in a bell-shapedway within a given range of zinc concentration. Also, the high zincconcentration effects observed by Ogunniyi et al {Ogunniyi 2009},leading to increased Pht expression, may have little in vivo relevancesince free zinc concentrations available in the human host are very low.

In 1997, Dintilhac et al {Dintilhac 1997a} concluded in their studythat, besides Psa, described as an ABC-type Mn²⁺ permease, and Adc, anABC-type Zn²⁺ permease, a third transporter should exist, capable oftransporting both Zn²⁺ and Mn²⁺. The Pht proteins or the laminin-bindingprotein would appear as candidates to fulfill this function. Our resultsare indicative of a different role for the Phts. Indeed, the fact thatwild-type and PhtD-deficient strains were able to grow in minimal mediumin the absence of Zn²⁺ and Mn²⁺ is intriguing. In addition, theobservation that the quadruple mutant growth was rescued with a delaywhen Zn²⁺ or Mn²⁺ was added to the minimal medium is also intriguing.These observations could be explained if we consider that the Phtproteins act as Zn²⁺ and Mn²⁺ scavengers, with the function to store andconcentrate those divalent cations. When wild-type and PhtD-deficientmutant strains were put into minimal medium, they were able to startgrowing immediately thanks to the ions stored previously within the Phtproteins when bacteria were in a richer medium. In contrast, thequadruple Pht-mutants were not able to store those ions when placed infavorable conditions, and then could not grow when put in poor medium.When Zn²⁺ or Mn²⁺ were added in excess to minimal medium, some time wasneeded before the ions could be caught by the specific metal permeases,because they had to find them at random in the culture medium, withouthelp from Pht proteins. This might explain the delay needed for thequadruple Pht mutant to start growing in such conditions. Moreover, apossible scavenging role for the Pht proteins is consistent with thepresence of five to six cation-binding domains.

This speculative mechanism of storage, if confirmed, could be consideredas a means for the bacterium to regulate zinc and probably manganesehomeostasis. Metal ions like zinc and manganese are essential traceelements. However, they are potentially harmful to the bacterium when inexcess, because they may compete with other elements as co-factors forsome critical enzymes. Therefore, it is essential for the bacteria toregulate metal homeostasis, and we suggest that this is the main role ofthe Pht family. Such a regulatory system would allow S. pneumoniae tosurvive when facing ion-restricted environments, for example during theinitial stages of the colonization process in human nasopharynx {Bunker1984, Harlyk 1997}.

The existence of polycistronic transcripts with PhtD might be explainedby the requirement of Zn²⁺ or Mn²⁺ for Lmb, an Lral family member, andYfnA, to exert their function. To partly support this statement, it hasbeen suggested that Mn²⁺ is required for adhesion through the Lralfamily of proteins, a critical feature for virulence {Dintilhac 1997b,Papp-Wallace 2006}. In addition, it has been demonstrated in othercontexts that laminin binds Zn²⁺ to promote high affinity bindingbetween laminin and laminin binding proteins {Ancsin 1996, Bandyopadhyay2002}. Therefore, we may hypothesize that Lmb needs PhtD to assure thepresence of Zn²⁺ when Lmb encounters laminin, which enhance binding tothe host tissues. The regulation of zinc homeostasis by the Phts mayalso explain why these proteins have been associated with the inhibitionof C3b (Hostetter, 1999 41/id; Ogunniyi, 2009 98/id). Indeed, thecleavage of C3b by factor I in the presence of factor H is regulated byzinc {Blom 2003}. By controlling zinc concentration in the bacterialenvironment, the Phts might thus contribute to C3b inhibition in somecircumstances, which needs to be investigated further.

Therefore, by targeting the Pht protein family, the immune system mayimpede the possibility for the bacteria to store and use ions, whichappear to be crucial for the invasion process. Consequently, our resultsconfirm the Pht proteins as genuine vaccine candidates againstpneumococcal infections. The different members of the Pht family havealready been evaluated for their potential to be used as pneumococcalvaccine antigens {Adamou 2001, Hamel 2004, Ogunniyi 2007, Zhang 2001}.After their discovery, PhtA, PhtB and PhtD were examined for theirability to protect mice against a subset of pneumococcal isolates{Adamou 2001}. PhtD was found to be the Pht protein that affords thebroadest protection, while PhtA immunization was efficient against alesser number of the strains tested. This is in line with the results ofthe present study, where it is shown that PhtA is expressed in 62% ofpneumococcal strains, while PhtD is present in 100%. Althoughsuccessfully used in two studies, the potential for PhtB to elicitcross-protection is not known since it was evaluated against a singlestrain only {Adamou 2001, Zhang 2001}.

However, since we found it in 81% of the strains, one may expect thatits inter-strain coverage might not be optimal. About PhtE, this proteinis found in 97% of the strains, which might be indicative of a broadcross-protection. However, this Pht shares only 32% of identity with thethree other Phts, and its C-terminal part, the most immunogenic andconserved one, is PhtE-specific. The region of PhtE common with theother Phts is not accessible to antibodies {Adamou 2001, Hamel 2004}.Therefore, PhtD, present in all strains tested, with an amino-acidsequence highly conserved among pneumococci and also demonstratingcross-reactivity with PhtA and PhtB, represents a better option.

Example 7 Immunization with Pht Proteins Confers Protection in a MousePneumococcal Lethal Intranasal Challenge Model

To evaluate the protection afforded by the members of the Pht family ina mouse pneumococcal intranasal challenge, OF1 female mice (four-weeksold; n=20/group) were immunized intramuscularly (i.m.) at day 0 and 14with 1 μg of PhtD, PhtA, PhtB, or PhtE, formulated with the AS02Adjuvant System that consists of an oil-in-water emulsion supplementedwith 3-O-desacyl-4′-monophosphoryl lipid A (MPL) and QS21 (Garcon et alExpert Rev. Vaccines 6; 723-739 (2007). Control animals were injectedwith AS02 only. At day 28, the mice were challenged intranasally withthe type 3/43 pneumococcal strain (10⁵ cfu in 50 μl). Mortality wasrecorded for 10 days after the challenge.

In other experiments, vaccination with 1 μg of PhtD was compared with 10μg of PspA and 10 μg of CbpA. All antigens were formulated with theAdjuvant System AS04, consisting of aluminum salts with MPL (Garcon etal Expert Rev. Vaccines 6; 723-739 (2007). The i.m. immunizationsoccurred at day 0, 14 and 28. Control animals were vaccinated withadjuvant only. At day 42, the mice were challenged intranasally with S.pneumoniae type 4/CDC (5×10⁶ cfu), type 2/D39 (2×10⁵ cfu), or type 3/43(10⁵ cfu) in 50 μl. The mortality was recorded for 10 days after thechallenge. Survival data were analyzed with the logrank test(Mantel-Haenszel).

The results indicate that vaccination with either of the Pht proteinsallowed the survival of approximately 60% of mice, while only 20% of theanimals survived in the control group (FIG. 1).

In subsequent experiments, other groups of animals were vaccinated withthree different pneumococcal antigens, PspA, CbpA, and one protein ofthe Pht family, namely PhtD. The extent of the humoral response wasevaluated and the animals were challenged with three differentpneumococcal strains two weeks after the last immunization. Micesurvival was recorded for all antigen/strain combinations. The resultinglevels of antibodies were antigen-dependent (FIG. 2A). Vaccination with1 μg of PhtD elicited higher antibody titers than vaccination with 10 μgof CbpA or PspA. Nevertheless, the level of protection againstintra-nasal lethal challenge with the 2/D39 strain, from which CbpA andPspA originate, was similar for the three antigens, with around 70% ofsurvival (FIG. 3A). Differences between the antigens were evidenced whenother strains were used. Indeed, vaccination with PhtD allowed 60% and80% of mice to survive the challenge with the 3/43 and the 4/CDCstrains, respectively. In contrast, CbpA and PspA afforded no or veryweak protection against the type-3 and -4 challenges. PhtD was thus theonly antigen able to afford protection against the three strains.

Example 8 Immunization with Pht Proteins Protects Mice Against S.Pneumoniae Nasopharyngeal Colonization

A nasopharyngeal colonisation assay was used to assess the ability ofimmunization against PhtD to prevent otitis media. Several studies haveshown a link between nasopharylgeal colonisation and otitis media.Bogaert et al Lancet Infect. Dis. 4(3); 144-154 (2004) showed thatcolonisation rates tend to be higher during respiratory tract infectionand otitis media. Indeed pneumococcal disease will not occur withoutproceeding and/or concurrent nasopharyngeal colonisation with thehomologous strain (Grey et al J. Infect. Dis. 142; 923-933 (1980),Syrjanea et al Paediatr. Infect. Dis. J. 24; 801-806 (2005)).

Balb/c mice (four-weeks old; n=10/group) were immunized at days 0, 14and 28 by the intranasal route with 5 μg of PhtD, PhtA, PhtB, or PhtEsupplemented with 0.2 μg of E. coli labile toxin (LT) as an adjuvant(except in the last immunization). Another experiment with the sameprotocol (schedule and dosages) consisted in comparing PhtD with CbpA,PsaA, and PspA. Control mice were injected with LT alone. At day 42,mice were challenged intranasally with 7×10⁴ cfu of type 6B/CDC strain,type 4/CDC, or type 2/D39. The challenges were performed using a smallbacterial inoculum volume (10 μl). Bacterial colonies were counted innasal washings collected 2 and 6 days after the challenge. Nasalwashings were obtained by flushing 500 μl of PBS inside the nasal cavityof anaesthetized mice. Next, to count the bacterial colonies, 100 μl ofnasal washing was diluted ten times in Todd Hewitt Broth. From this, 10μl was plated onto Difco™ Blood Agar base supplemented withdefinibrated, sterile sheep blood and gentamycin (3 μg/ml). The Petridish was tilted to spread the sample and the colonies were enumeratedafter incubation overnight at 37° C. All colony countings data, afternormalization, were compared with ANOVA, followed by the Dunnettpost-test when ANOVA was found significant.

To assess the protective activity of vaccination against naso-pharyngealcarriage, Balb/c mice were immunized intranasally with the different Phtproteins before they were challenged via the same route with the 2/D39strain. As can be seen in FIG. 4, although only vaccination with PhtD orPhtE afforded significant protection against the challenge with the type2 strain, all members of the Pht family were able to reduce bacterialload in the nasopharynx of the vaccinated animals. Due to the betterperformance of PhtD in this model, this member of the Pht family waschosen for further experiments, consisting in comparing PhtD with otherpneumococcal proteins.

Therefore, mice were immunized with different pneumococcal antigens,including PhtD, and were subsequently challenged with a type 2, a type 4or a type 6B strain. As was observed after systemic immunization, theelicited humoral responses after intra-nasal immunization wereantigen-dependent (FIG. 2B). Particularly, CbpA elicited lower antibodytiters than PspA and PhtD. However, the level of protection afforded byCbpA against the clade-homologous 2/D39 strain was similar to that ofPspA and PhtD (FIG. 5A).

When the type4/CDC was used for the challenge, only immunization withPhtD could protect the animals against naso-pharyngeal colonization,whereas immunization with CbpA, PsaA or PspA was not statisticallydistinguishable from the LT control (FIG. 5B). Finally, challenge withtype 6B/CDC did not evidence any difference in protection at day 2post-challenge whether the animals were immunized with CbpA, PspA orPhtD (FIG. 5C). Only PsaA seemed to be less efficient in that respect.At day 6 post-challenge, there was no statistical difference between allgroups. However, a careful examination of the results for PhtD revealedthat the majority of animals were protected against naso-pharyngealcolonization and that the unfavorable statistical conclusion wasprobably only due to the presence of two outliers. In conclusion, PhtDwas the only antigen able to afford some protection against the threestrains in this model of naso-pharyngeal colonization.

Example 9 Immunization with PhtD Protects Mice Against S. PneumoniaeLung Colonization

The model was adapted from Briles et al J. Infect. Dis. 188; 339-348(2003). CBA/J female mice (four-weeks old; 30/group) were immunized i.m.at day 0, 14 and 28 with 3 μg of PhtD adjuvanted with AS02. At day 42,the mice were challenged intranasally with 2×10⁷ cfu/50 μl of S.pneumoniae 19F/2737. Control mice were injected with adjuvant only.Bacterial load was measured by colony counting in lungs collected 3, 4and 5 days post-challenge. All colony countings data, afternormalization, were compared with ANOVA, followed by the Dunnettpost-test when ANOVA was found significant.

CBA/J mice, a strain susceptible to pneumococcal infections, werevaccinated with PhtD before they were challenged with a moderatelyvirulent 19F bacterial strain. Such a protocol allows for the inductionof a focal pneumonia without generalized sepsis. After the challenge,the number of living bacteria in the lungs was evaluated at day 3, 4 and5.

It was shown that vaccination with PhtD reduced the bacterial load inthe lungs to a great extent (more than 95%), as compared with placebo(FIG. 6 a). The efficacy of PhtD vaccination was particularly evidentwhen analyzing the number of non-colonized mice, since up to 80% ofvaccinated mice remained free of bacteria at day 5, as compared with 10%in the control group (FIG. 6 b).

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1.-75. (canceled)
 76. A method of treating a Streptococcus pneumoniaeinfection in a mammal comprising the step of administering apharmaceutically effective amount of at least one PhtX protein to themammal.
 77. The method of treatment of claim 76 wherein the PhtX proteinis selected from the group consisting of PhtA, PhtB, PhtD and PhtE. 78.The method of claim 76 wherein the PhtX protein is PhtD.
 79. The methodof claim 76 wherein the Streptococcus pneumoniae infection occurs in alung.
 80. The method of claim 76 wherein the Streptococcus pneumoniaeinfection occurs in a lung and the treatment reduces the bacterial loadin the lungs.
 81. The method of claim 76 wherein the Streptococcuspneumoniae infection occurs in a middle ear.
 82. The method of claim 76wherein the mammal is a human.
 83. The method of claim 76 wherein theStreptococcus pneumoniae infection is septicaemia, bacteraemia,meningitis, otitis media or pneumonia.
 84. An immunogenic compositioncomprising a pharmaceutically effective amount of an isolated PhtXprotein for use in the treatment of a Streptococcus pneumoniae infectionwherein the Streptococcus pneumoniae infection occurs in a mammal in anenvironment where the free concentration of Zn²⁺ and/or Mn²⁺ issufficiently low to upregulate the expression of at least one PhtXprotein in the Streptococcus pneumoniae.
 85. The immunogenic compositionof claim 84 wherein the PhtX protein is selected from the groupconsisting of PhtA, PhtB, PhtD and PhtE.
 86. The immunogenic compositionof claim 85 wherein the PhtX protein is PhtD.
 87. The immunogeniccomposition of claim 84 wherein the Streptococcus pneumoniae infectionoccurs in a lung.
 88. The immunogenic composition of claim 84 whereinthe Streptococcus pneumoniae infection occurs in a middle ear.
 89. Theimmunogenic composition of claim 84 wherein the Streptococcus pneumoniaeinfection is septicaemia, bacteraemia, meningitis, otitis media orpneumonia.